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Título: | Combinatorial saturation mutagenesis of the Myceliophthora thermophila laccase T2 mutant: The connection between the C-terminal plug and the conserved509VSG511 tripeptide |
Autor: | Zumárraga, Miren CSIC; Vaz Domínguez, C. CSIC; Camarero, Susana CSIC ORCID ; Shleev, Sergey; Polaina Molina, Julio CSIC ORCID; Martínez Arias, Arturo CSIC ORCID; Ferrer, Manuel CSIC ORCID; López de Lacey, Antonio CSIC ORCID ; Fernández López, Víctor Manuel; Ballesteros Olmo, Antonio CSIC ORCID; Plou Gasca, Francisco José CSIC ORCID ; Alcalde Galeote, Miguel CSIC ORCID | Palabras clave: | combinatorial saturation mutagenesis Ascomycete laccases C-terminal plug redox potential Saccharomyces cerevisiae |
Fecha de publicación: | 2008 | Editor: | Bentham Science Publishers | Citación: | Combinatorial Chemistry and High Throughput Screening 11: 807- 816 (2008) | Resumen: | A mutant laccase from the Ascomycete Myceliophthora thermophila has been submitted to iterative cycles of combinatorial saturation mutagenesis through in vivo overlap extension in Saccharomyces cerevisiae. Over 180,000 clones were explored, among which the S510G mutant revealed a direct interaction between the conserved 509VSG511 tripeptide, located in the neighborhood of the T1 site, and the C-terminal plug. The Km O 2 value of the mutant increased 1.5-fold, and the electron transfer pathway between the reducing substrate and the T1 copper ion was altered, improving the catalytic efficiency towards non-phenolic and phenolic substrates by about 3- and 8-fold. Although the geometry at the T1 site was perturbed by the mutation, paradoxically the laccase redox potential was not significantly altered. Together, the results obtained in this study suggest that the 509VSG511 tripeptide may play a hitherto unrecognized role in regulating the traffic of oxygen through the C-terminal plug, the latter blocking access to the T2/T3 copper cluster in the native enzyme. | Versión del editor: | https://doi.org/10.2174/138620708786734235 | URI: | http://hdl.handle.net/10261/191607 | DOI: | 10.2174/138620708786734235 | Identificadores: | doi: 10.2174/138620708786734235 issn: 1386-2073 e-issn: 1875-5402 |
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